This study investigates the interaction of actin with the subfragments of myosin, subfragment-one (S-1) and heavy meromyosin (HMM), in order to define the role of the two heads of myosin in muscle contraction. The association constant for the binding of the single-headed S-1 fragment to actin was measured both in the presence and absence of nucleotide. The association constant for S-1 binding to actin is 5 times 10 to the 6th power M minus 1 at micron equals 0.22M, 22 degrees. This binding becomes approximately 50-fold weaker in the presence of ADP, and 500-fold weaker in the presence of AMP-PNP or PPi. The ability of these ATP analogs to dissociate acto S-1 enables states in the crossbridge cycle to be studied which occur only transiently when ATP is present. In addition, this study examines the binding of the two-headed HMM fragment to actin to determine how the two heads of myosin interact. In the absence of nucleotides, HMM binds to actin with a stoichiometry of one HMM per two F-actin monomers and an association constant of 2 times 10 to the 9th power M minus 1. This association constant is 400-fold stronger than the acto S-1 association constant. In the presence of AMP-PNP, HMM binds to actin with only a two-fold greater association constand than S-1, indicating that HMM may be binding to actin with only one head.